Evidence for a role of human organic anion transporters in the muscular side effects of HMG-CoA reductase inhibitors

https://doi.org/10.1016/j.ejphar.2003.10.017Get rights and content

Abstract

The purpose of this study was to elucidate the role of human organic anion transporters (human OATs) in the induction of drug-induced skeletal muscle abnormalities. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have been clinically used for lowering plasma cholesterol levels, and are known to induce various forms of skeletal muscle abnormalities including myopathy and rhabdomyolysis. Immunohistochemical analysis revealed that human OAT1 and human OAT3 are localized in the cytoplasmic membrane of the human skeletal muscles. The activities of human OATs were measured using mouse cell lines from renal proximal tubules stably expressing human OATs. Human OAT3, but not human OAT1, mediates the transport of pravastatin. Fluvastatin inhibited organic anion uptake mediated by human OAT1 in a mixture of competitive and noncompetitive manner, whereas simvastatin and fluvastatin noncompetitively inhibited the organic anion uptake mediated by human OAT3. In conclusion, the organic anion transporters OAT1 and OAT3 are localized in the cytoplasmic membrane of human skeletal muscles. Pravastatin, simvasatin, and fluvasatin inhibit human OATs activity. These results suggest that muscle organic anion transporters play a role in the muscular side effects of HMG-CoA reductase inhibitors.

Introduction

The excretion of numerous organic anions, including endogenous metabolites, drugs, and xenobiotics, is an important physiological function of the kidney. Recently, cDNAs encoding the human organic anion transporters (human OATs) have been successively cloned, including human OAT1 Reid et al., 1998, Hosoyamada et al., 1999, human OAT2 (Enomoto et al., 2002), human OAT3 (Cha et al., 2001), and human OAT4 (Cha et al., 2000). We have been elucidating the localization and functional roles of human OATs mainly in the kidney.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is the rate-limiting step in cholesterol biosynthesis from acetate. The inhibition of this enzyme reduces cytoplasmic cholesterol levels in hepatocytes, which respond by increasing the synthesis of low-density lipoprotein (LDL) receptors that are expressed on hepatocyte surface membranes. This in turn increases hepatic LDL uptake from the plasma, reducing the plasma LDL concentration (Ritter et al., 1999). Thus, HMG-CoA reductase inhibitors have been used to lower plasma total and LOL levels in patients with hypercholesterolemia, which prevents the progression of atherosclerosis.

It has been recently shown that pravastatin, a HMG-CoA reductase inhibitor that possesses anionic moieties, is transported by rat OAT1 and rat OAT3 (Hasegawa et al., 2002). In addition to the kidney, mRNAs from human OATs were shown to be expressed in various organs including the brain, liver, skeletal muscles, and placenta Hosoyamada et al., 1999, Cha et al., 2000, Cha et al., 2001. In the human skeletal muscles, HMG-CoA reductase inhibitors, including pravastatin, fluvastatin, and simvastatin (Fig. 1), were shown to induce various forms of skeletal muscle abnormalities ranging from mild myopathy to myositis, and occasionally rhabdomyolysis and even death (Evans and Rees, 2002). These lines of evidence raise the possibility that human OAT1 and human OAT3 localized in the cytoplasmic membrane of the skeletal muscles mediate the uptake of HMG-CoA reductase inhibitors into the skeletal muscles and the induction of skeletal muscle abnormalities. In order to elucidate this hypothesis, firstly, we performed an immunohistochermical analysis of human OAT1 and human OAT3 in the human skeletal muscles. Secondly, we examined whether human OAT1 and human OAT3 mediate the transport of pravastatin. Thirdly, we elucidated the interactions of human OAT1 and human OAT3 with HMG-CoA reductase inhibitors, including fluvastatin and simvastatin. The activities of human OATs were measured using mouse cell lines from renal proximal tubules stably expressing human OATs.

Section snippets

Materials

Adult human normal skeletal muscle tissue slides were purchased from Bio-chain (Hayward, CA, USA). The human skeletal muscle tissues were from a 26-year-old male. Other materials used included fetal bovine serum, trypsin, and geneticin from Invitrogen (Carlsbad, CA); recombinant epidermal growth factor from Wakunaga (Hiroshima, Japan); insulin from Shimizu (Shizuoka, Japan); RITC 80-7 culture medium from Iwaki (Tokyo, Japan); and simvastatin from Wako (Tokyo, Japan). [14C]pravastatin and

Immunolocalization of human OAT1 and human OAT3 in human skeletal muscles

As shown in Fig. 2, human OAT1 (A) and human OAT3 (B) were stained in the cytoplasmic membrane of the skeletal muscles, but the muscle fibers were not stained. The negative control exhibited no immunoreactivity (C).

Pravastatin uptake mediated by human OAT1 and human OAT3

We examined whether human OAT1 and human OAT3 mediate the uptake of pravastatin. As shown in Fig. 3, human OAT3, but not human OAT1, exhibited significantly higher uptake activities of pravastatin than mock cells (n=8, *P<0.001 vs. mock).

Effects of fluvastatin and simvastatin on organic anion uptake mediated by human OAT1 and human OAT3

We examined the inhibitory effects of various

Discussion

Among adverse drug reactions by HMG-CoA reductase inhibitors, skeletal muscle abnormalities and hepatotoxicity are the most clinically important (Omar et al., 2001). Increases of creatine kinase concentrations to more than three times the upper limit of normal have been reported in 3–5% of patients Omar et al., 2001, Stalenhoef et al., 1989, Ziegler and Drouin, 1990. Skeletal muscle abnormalities can range from benign myalgia to myopathy, which is defined as a 10-fold elevation of the creatine

Acknowledgments

This study was supported, in part, by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology (nos. 11671048, 11694310, 13671128, and 15590858), the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and the fund for Research on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation.

References (24)

  • S.H. Cha et al.

    Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta

    J. Biol. Chem.

    (2000)
  • L.J. Leventhal et al.

    Salicylate-induced rhabdomyolysis

    Am. J. Emerg. Med.

    (1989)
  • Y. Berland et al.

    Rhabdomyolysis with simvastatin use

    Nephron

    (1991)
  • S.H. Cha et al.

    Identification and characterization of human organic anion transporter 3 expressing predominantly in the kidney

    Mol. Pharmacol.

    (2001)
  • J.G. Dain et al.

    Biotransformation of fluvastatin sodium in humans

    Drug Metab. Dispos.

    (1993)
  • A. Enomoto et al.

    Interaction of human organic anion transporters 2 and 4 with organic anion transport inhibitors

    J. Pharmacol. Exp. Ther.

    (2002)
  • M. Evans et al.

    Effects of HMG-CoA reductase inhibitors on skeletal muscle

    Drug Safety

    (2002)
  • W.R. Garnett

    Interactions with hydroxymethylglutaryl-coenzyme A reductase inhibitors

    Am. J. Health Syst. Pharm.

    (1995)
  • J.G. Hardman et al.

    The Pharmacological Basis of Therapeutics

    (2001)
  • M. Hasegawa et al.

    Functional involvement of rat organic anion transporter 3 (rOat3; Slc22a8) in the renal uptake of organic anions

    J. Pharmacol. Exp. Ther.

    (2002)
  • R. Herman

    Drug interactions and the statins

    CMAJ.

    (1999)
  • M. Hosoyamada et al.

    Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney

    Am. J. Physiol.

    (1999)
  • Cited by (85)

    • Investigating the interaction between organic anion transporter 1 and ochratoxin A: An in silico structural study to depict early molecular events of substrate recruitment and the impact of single point mutations

      2022, Toxicology Letters
      Citation Excerpt :

      As reported in the USCD-FDA TransPortal database, all these ligands have been already described as high affinity substrates of OAT1 with Km values in the low μM range (Cihlar and Ho, 2000; Islinger et al., 2001; Jung et al., 2001; Kimura et al., 2002). The set of decoys was collected from the scientific literature and included homostachydrine, glycitein-7-glucuronide, pravastatin and beta-muricholate (Fig. 3) as previous studies ascertained that they are not appreciably transported or bound by OAT1 (Bush et al., 2017; Takeda et al., 2004; Wong et al., 2011). Of note, the docking software used in this study used a scoring function that provides an estimate of the binding energy (i.e. the more negative the score, the more stable the expected stability of the protein-ligand complex).

    • Interactions of human organic anion transporters 1–4 and human organic cation transporters 1–3 with the stimulant drug methamphetamine and amphetamine

      2020, Legal Medicine
      Citation Excerpt :

      We examined seven different types of transporters that mediate the transport of drugs into cells. cDNAs have been successively cloned that encode the organic anion transporters (OATs) OAT1 [5–9], OAT2 [10–13], OAT3 [14–17], and OAT4 [18–20], and their sites of localization have been elucidated. Whereas hOAT1, hOAT3, and hOAT4 are primarily expressed in the kidney, hOAT2 is primarily expressed in the liver.

    • Drug development

      2020, Metabolomics for Biomedical Research
    • Pemetrexed nephrotoxicity

      2015, Bulletin du Cancer
    View all citing articles on Scopus
    View full text